DESCRIPTION: Current approaches to transfer genes in vivo employ either recombinant viral vectors or non-viral delivery systems. In this project, the molecular biology of the human parvovirus adeno-associated virus (AAV) is studied with the intent to use the virion shell as a platform for developing a novel, safe, and efficient delivery system for human gene therapy. Previous research by this group uncovered a rate limiting step for AAV vector transduction; that is, the conversion of single stranded AAV to double stranded. In addition, the finite packaging capacity of this virus as restricted its use as a vector to small genes or cDNAs. In addition, virions that specifically and efficiently target defined cell types without transducing others will be required for clinical application. The long-term objective of the research is to develop novel delivery systems that exploit the advantages of AAV viral infectivity without the disadvantages of packaging constraints, rate limiting steps in second-strand synthesis, or the inability to target specific cell types Three approaches to address these issues will be analyzed in this proposal. In specific aim 1, domain(s) in the AAV2 capsid responsible for wild type (wt) receptor mediated uptake will be genetically identified and characterized. Targeted capsid mutants will be used to define the region of AAV2 required for receptor-mediated uptake. The mutant virions generated will be tested for encapsidation of vector DNA and virion assembly but defective for AAV-2 viral transduction. Columns carrying serotype 2 receptor will be used to determine if wild-type AAV-2 binding is lost. Identification of wt virions which are receptor minus mutants will be utilized when testing targeting vectors. In specific aim 2, AAV helper plasmids will be constructed for production of hybrid virions that maintain AAV infectivity and parvovirus B19 larger packaging capacity. Chimeric helper plasmids carrying AAV/B19 coding sequences will be constructed. Primarily, Vp2 of B19 will be swapped for AAV Vp3, and assayed for the packing of AAV-2 reporter vectors. Viable chimeric virions will be assayed for the ability to package larger genomes. Six vectors with increasing insertions from 745 to 1811 of additional bases (total 6.4 kb) are available which can be used to determine packaging capacity and the transduction capabilities of each vector will be tested. In specific aim 3, a novel Ad vector that expresses the AAV capsid will be characterized for production of empty particles and virion DNA conjugates will be tested for efficient in vitro and in vivo delivery. Empty AAV particles will be characterized for binding and uptake on target cells using fluorescent-labeled virions and confocal microscopy. Empty particles will be conjugated with AAV reporter plasmid vectors (PacZ and GFP) and tested for efficient gene delivery in vitro. Viable AAV receptor-minus mutants, chimeric particles, or targeted virions identified in specific aims 1 and 2 will be engineered into the Ad/AAV capsid expression vector for over-production of modified empty virions. Empty particles carrying specific alterations will be assayed for infectivity and gene delivery in vitro and in vivo.